Composition for reducing nervous system injury and method of making and use thereof

ABSTRACT

This application discloses a composition comprising an amiloride and/or an amiloride analog which can be used for reducing nerve injury or nervous system injury in a subject. The formulation of such composition is also disclosed. The application further directs to methods for treating nerve injury or nervous system injury by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog or a pharmaceutically acceptable salt thereof.

This application is a continuation of U.S. patent application Ser. No.13/972,558, filed on Aug. 21, 2013, which is a continuation-in-part ofU.S. patent application Ser. No. 13/284,166, filed on Oct. 28, 2011,which is a continuation of U.S. patent application Ser. No. 11/724,859,filed Mar. 16, 2007, now U.S. Pat. No. 8,076,450, which claims priorityto U.S. Provisional Patent Application No. 60/611,241, filed Sep. 16,2004. The entirety of the aforementioned applications is incorporatedherein by reference.

FIELD

This application relates to the field of neurology. In particular, thisapplication directs to compositions comprising an amiloride and/or anamiloride analog which can be used for reducing nerve injury or nervoussystem injury in a subject.

BACKGROUND

Nerve injuries may be caused by many conditions, such as degenerativenerve diseases, stroke, ischemia, chemical and mechanical injury to thenervous system. Many types of nerve injury result in changes in the ionflux into neurons which, in turn, lead to neuron cell death.Accordingly, various ion channels may be candidates for mediating thisaltered ion flux, thus reducing the extent of nerve injuries.

SUMMARY

One aspect of the present application relates to a method for reducingnerve injury in a subject. The method comprises administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising an active ingredient selected from the groupconsisting of amiloride and amiloride analogs. In some embodiments, thepharmaceutical composition is administered intravenously, intrathecallyor intracerebroventricularlly.

In some embodiments, the active ingredient comprises amiloride or apharmaceutically acceptable salt thereof

In other embodiments, the active ingredient comprises an amilorideanalog or a pharmaceutically acceptable salt thereof. In a relatedembodiment, the amiloride analog is selected from the group consistingof benzamil, phenmil, 5-(N-ethyl-N-isobutyl)-amiloride (EIPA), bepridil,KB-R7943, 5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimenthyl) amiloride hydrochloride. In anotherrelated embodiment, the amiloride analog is benzamil. In another relatedembodiment, the amiloride analog is a methylated analog of benzamil. Inanother related embodiment, the amiloride analog comprises a ring formedon a guanidine group. In another related embodiment, the amilorideanalog comprises an acylguanidino group. In another related embodiment,the amiloride analog comprises a water solubilizing group formed on aguanidine group, wherein the water solubilizing group is a N,N-dimethylamino group or a sugar group.

In some embodiments, the amiloride, amiloride analog or apharmaceutically acceptable salt thereof is given in a dose range of 0.1mg-10 mg/kg body weight.

In some embodiments, the pharmaceutical composition is administeredwithin one hour of the onset of an ischemic event, within five hours ofthe onset of an ischemic event, or between one hour and five hours ofthe onset of an ischemic event.

In some embodiments, the nerve injury is brain injury.

Another aspect of the present application relates to a method fortreating brain injury in a subject. The method comprises administeringto said subject a therapeutically effective amount of a pharmaceuticalcomposition comprising amiloride, an amiloride analog, or apharmaceutically acceptable salt thereof. In some embodiments, thepharmaceutical composition is administered intravenously, intrathecallyor intracerebroventricularly.

In some embodiments, the amiloride analog is selected from the groupconsisting of benzamil, phenmil, EIPA bepridil, KB-R7943,5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and5-(N,N-dimenthyl) amiloride hydrochloride. In a related embodiment, theamiloride analog is benzamil. In other embodiments, the amiloride analogis selected from the group consisting of methylated analogs of benzamil,amiloride analogs containing a ring formed on a guanidine group,amiloride analogs containing an acylguanidino group, and amilorideanalogs containing a water solubilizing group formed on a guanidinegroup, wherein the water solubilizing group is a N,N-dimethyl aminogroup or a sugar group.

Another aspect of the present application relates to a method forreducing nervous system injury caused by a change of ion flux intoneurons. The method comprises administering to a subject in need of suchtreatment a therapeutically effective amount of a pharmaceuticalcomposition comprising amiloride, an amiloride analog or apharmaceutically acceptable salt thereof

In some embodiments, the pharmaceutical composition is administeredintravenously, intrathecally, intracerebroventricularly orintramuscularly.

In other embodiments, the amiloride analog is selected from the groupconsisting of benzamil, phenmil, EIPA bepridil, KB-R7943,5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and5-(N,N-dimenthyl) amiloride hydrochloride. In a related embodiment, theamiloride analog is benzamil. In other embodiments, the amiloride analogis selected from the group consisting of methylated analogs of benzamil,amiloride analogs containing a ring formed on a guanidine group,amiloride analogs containing an acylguanidino group, and amilorideanalogs containing a water solubilizing group formed on a guanidinegroup, wherein the water solubilizing group is a N,N-dimethyl aminogroup or a sugar group.

Another aspect of the present application relates to a method forreducing nervous system injury. The method comprises administering to asubject in need of such treatment a therapeutically effective amount ofa pharmaceutical composition comprising amiloride, an amiloride analogor a pharmaceutically acceptable salt thereof

In some embodiments, the pharmaceutical composition is administeredintravenously, intrathecally, intracerebroventricularly orintramuscularly.

In other embodiments, the amiloride analog is selected from the groupconsisting of benzamil, phenmil, EIPA bepridil, KB-R7943,5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and5-(N,N-dimenthyl) amiloride hydrochloride. In a related embodiment, theamiloride analog is benzamil. In other embodiments, the amiloride analogis selected from the group consisting of methylated analogs of benzamil,amiloride analogs containing a ring formed on a guanidine group,amiloride analogs containing an acylguanidino group, and amilorideanalogs containing a water solubilizing group formed on a guanidinegroup, wherein the water solubilizing group is a N,N-dimethyl aminogroup or a sugar group.

Another aspect of the present application relates to a pharmaceuticalcomposition for reducing nervous system injury. The pharmaceuticalcomposition comprises an effective amount of amiloride, an amilorideanalog or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated for intravenous, intrathecal orintracerebroventricular injection.

In some embodiments, the pharmaceutical composition comprises anamiloride analog or a pharmaceutically acceptable salt thereof, whereinthe amiloride analog is selected from the group consisting of benzamil,phenmil, EIPA bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride,5-(N,N-hexamethylene) amiloride and 5-(N,N-dimenthyl) amiloridehydrochloride.

In other embodiments, the pharmaceutical composition comprises anamiloride analog or a pharmaceutically acceptable salt thereof, whereinthe amiloride analog is selected from the group consisting of methylatedanalogs of benzamil, amiloride analogs containing a ring formed on aguanidine group, amiloride analogs containing an acylguanidino group,and amiloride analogs containing a water solubilizing group formed on aguanidine group, wherein the water solubilizing group is a N,N-dimethylamino group or a sugar group.

Another aspect of the present application relates to a pharmaceuticalcomposition for reducing nervous system injury. The pharmaceuticalcomposition comprises an effective amount of an amiloride analog or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.

In some embodiments, the pharmaceutical composition is formulated forintravenous, intrathecal, intracerebroventricular or intramuscularinjection.

In one embodiment, the amiloride analog is a methylated analog ofbenzamil. In another embodiment, the amiloride analog comprises a ringformed on a guanidine group. In another embodiment, the amiloride analogcomprises an acylguanidino group. In yet another embodiment, theamiloride analog comprises a water solubilizing group formed on aguanidine group, wherein the water solubilizing group is a N,N-dimethylamino group or a sugar group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of a flowchart illustrating an exemplary method ofreducing neuroinjury in an ischemic subject.

FIG. 2 is a view of a flowchart illustrating an exemplary method ofidentifying drugs for treating ischemia-related nerve injury.

FIGS. 3A-D are a series of graphs presenting exemplary data related tothe electrophysiology and pharmacology of acid sensing ion channel(ASIC) proteins in cultured mouse cortical neurons.

FIGS. 4A-D are an additional series of graphs presenting exemplary datarelated to the electrophysiology and pharmacology of ASIC proteins incultured mouse cortical neurons.

FIGS. 5A-D are a set of graphs and traces presenting exemplary datashowing that modeled ischemia may enhance activity of ASIC proteins, inaccordance with aspects of the present teachings.

FIGS. 6A-B and 7A-D are a set of graphs and traces presenting exemplarydata showing that ASIC proteins in cortical neurons may be Ca²⁺permeable, and that Ca²⁺ permeability may be ASIC1a dependent.

FIGS. 8A-C are a series of graphs presenting exemplary data showing thatacid incubation may induce glutamate receptor-independent neuronalinjury that is protected by ASIC blockade.

FIGS. 9A-D are a series of graphs presenting exemplary data showing thatASIC1a may be involved in acid-induced injury in vitro.

FIGS. 10A-D are a series of graphs with data showing neuroprotection inbrain ischemia in vivo by ASIC1a blockade and by ASIC1 gene knockout.

FIG. 11 is a graph plotting exemplary data for the percentage ofischemic damage produced by stroke in an animal model system as afunction of the time and type of treatment.

FIG. 12 is a view of the primary amino acid sequence of an exemplarycystine knot peptide, PcTx1, with various exemplary peptide featuresshown.

FIG. 13 is a comparative view of the cystine knot peptide of FIG. 12aligned with various exemplary deletion derivatives of the peptide.

FIG. 14 is an exemplary graph plotting the amplitude of calcium currentmeasured in cells as a function of the ASIC family member(s) expressedin the cells.

FIG. 15 is a graph presenting exemplary data related to the efficacy ofnasally administered PcTx venom in reducing ischemic injury in an animalmodel system.

FIGS. 16A-C are a composite showing representative ASIC la currenttraces in CHO cells treated with benzamil (panel A) or5-(N-ethyl-N-isopropyl) amiloride (EIPA) (panel B), and dose-dependentblockade of ASIC la current expressed in CHO cells by amiloride andamiloride analogs (panel C).

FIGS. 17A-C are a composite showing representative ASIC 2a currenttraces in CHO cells treated with benzamil (panel A) or amiloride (panelB), and dose-dependent blockade of ASIC 2a current expressed in CHOcells by amiloride and amiloride analogs (panel C).

FIG. 18 is a graph showing reduction of infarct volume in mice byintracerebroventricular injections of amiloride or amiloride analogs.

FIG. 19 is a composite showing reduction of infarct volume in thecortical tissue of mice by intravenous injection of saline or amiloride60 min after MCAO.

FIG. 20 is a composite showing reduction of infarct volume in thecortical tissue of mice by intravenous injection of saline or amiloride3 hours or 5 hours after MCAO.

FIG. 21 shows structure activity relationship (SAR) for hydrophobicamiloride analogs on various channels.

DETAILED DESCRIPTION

The present application provides methods and compositions for reducingnerve injury. The nerve injury may be caused by degenerative nervoussystem diseases, stroke, ischemia, trauma, chemical and mechanicalinjury to the nervous system. As used herein, the term “nervous system”includes both the central nervous system and the peripheral nervoussystem.” The term “central nervous system” or “CNS” includes all cellsand tissue of the brain and spinal cord of a vertebrate. The term“peripheral nervous system” refers to all cells and tissue of theportion of the nervous system outside the brain and spinal cord, such asthe motor neurons that mediate voluntary movement, the autonomic nervoussystem that includes the sympathetic nervous system and theparasympathetic nervous system and regulates involuntary functions, andthe enteric nervous system that controls the gastrointestinal system.Thus, the term “nervous system” includes, but is not limited to,neuronal cells, glial cells, astrocytes, cells in the cerebrospinalfluid (CSF), cells in the interstitial spaces, cells in the protectivecoverings of the spinal cord, epidural cells (i.e., cells outside of thedura mater), cells in non-neural tissues adjacent to or in contact withor innervated by neural tissue, cells in the epineurium, perineurium,endoneurium, funiculi, fasciculi, and the like.

In some embodiments, the nerve injury is a nervous system injury. Inother embodiments, the nerve injury is brain injury. In someembodiments, the nerve injury is a nervous system injury caused bychanges in the ion flux into neurons. For example, stroke/brain ischemiais a leading cause of morbidity and mortality. Over-activation of thepostsynaptic glutamate receptors and subsequent Ca²⁺ toxicity plays acritical role in ischemic brain injury. The present applicationdemonstrates that activation of Ca²⁺-permeable acid-sensing ion channels(ASICs) is involved in acidosis-induced, glutamate receptor-independent,ischemic brain injury and provides a new direction for neuroprotectionby targeting ASIC family members (ASICs). The present applicationfurther provides novel inhibitors of ASICs that have increased potencyto homomeric ASICs channel and increased aqueous solubility. In someembodiments, the present application provides pharmaceuticalcompositions and methods for reducing nerve injury by inhibiting ASIC1achannel.

One aspect of the present application relates to a method for reducingnerve injury in a subject. The method comprises administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising an active ingredient selected from the groupconsisting of amiloride and amiloride analogs. In some embodiments, thepharmaceutical composition is administered intravenously, intrathecallyor intracerebroventricularlly.

In some embodiments, the active ingredient comprises amiloride or apharmaceutically acceptable salt thereof. In other embodiments, theactive ingredient comprises an amiloride analog or a pharmaceuticallyacceptable salt thereof. In a related embodiment, the amiloride analogis selected from the group consisting of benzamil, phenmil,5-(N-ethyl-N-isobutyl)-amiloride (EIPA), bepridil, KB-R7943,5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and5-(N,N-dimenthyl) amiloride hydrochloride. In another relatedembodiment, the amiloride analog is benzamil. In another relatedembodiment, the amiloride analog is a methylated analog of benzamil. Inanother related embodiment, the amiloride analog comprises a ring formedon a guanidine group. In another related embodiment, the amilorideanalog comprises an acylguanidino group. In another related embodiment,the amiloride analog comprises a water solubilizing group formed on aguanidine group, wherein the water solubilizing group is a N,N-dimethylamino group or a sugar group.

In some embodiments, the amiloride, amiloride analog or apharmaceutically acceptable salt thereof is given in a dose range of 0.1mg-10 mg/kg body weight. In other embodiments, the pharmaceuticalcomposition is administered within one hour of the onset of an ischemicevent, within five hours of the onset of an ischemic event, or betweenone hour and five hours of the onset of an ischemic event.

Another aspect of the present application relates to a method fortreating brain injury in a subject. The method comprises administeringto said subject a therapeutically effective amount of a pharmaceuticalcomposition comprising amiloride, an amiloride analog, or apharmaceutically acceptable salt thereof. In some embodiments, thepharmaceutical composition is administered intravenously, intrathecallyor intracerebroventricularly.

In some embodiments, the amiloride analog is selected from the groupconsisting of benzamil, phenmil, EIPA bepridil, KB-R7943,5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and5-(N,N-dimenthyl) amiloride hydrochloride. In a related embodiment, theamiloride analog is benzamil. In other embodiments, the amiloride analogis selected from the group consisting of methylated analogs of benzamil,amiloride analogs containing a ring formed on a guanidine group,amiloride analogs containing an acylguanidino group, and amilorideanalogs containing a water solubilizing group formed on a guanidinegroup, wherein the water solubilizing group is a N,N-dimethyl aminogroup or a sugar group.

Another aspect of the present application relates to a method forreducing nervous system injury caused by a change of ion flux intoneurons. The method comprises administering to a subject in need of suchtreatment a therapeutically effective amount of a pharmaceuticalcomposition comprising amiloride, an amiloride analog or apharmaceutically acceptable salt thereof

Another aspect of the present application provides a composition fortreating ischemia or reducing injury resulting from ischemia. The methodcomprises the step of administering intravenously or intrathecally to asubject in need of such treatment a therapeutically effective amount ofan active ingredient selected from the group consisting of amiloride,amiloride analogs, and salts thereof. The methods of the presentapplication may provide one or more advantages over other methods ofischemia treatment. These advantages may include (1) lessischemia-induced injury, (2) fewer side effects of treatment (e.g., dueto selection of a more specific therapeutic target), and/or (3) a longertime window for effective treatment, among others.

FIG. 1 shows a flowchart 20 with exemplary steps 22, 24 that may beperformed in a method of reducing nerve injury in an ischemic subject.The steps may be performed any suitable number of times and in anysuitable combination. In the method, an ischemic subject (or subjects)may be selected for treatment, indicated at 22. An ASIC-selectiveinhibitor then may be administered to the ischemic subject(s), indicatedat 24. Administration of the inhibitor to the ischemic subject may be ina therapeutically effect amount, to reduce ischemia-induced injury tothe subject, for example, reducing the amount of brain damage resultingfrom a stroke.

A potential explanation for the efficacy of the ischemia treatment ofFIG. 1 may be offered by the data of the present teachings (e.g., seeExample 1). In particular, the damaging effects of ischemia may not beequal to acidosis, that is, acidification of tissue/cells via ischemiamay not be sufficient to produce ischemia-induced injury. Instead,ischemia-induced injury may be caused, in many cases, by calcium fluxinto cells mediated by a member(s) of the ASIC family, particularlyASIC1a. Accordingly, selective inhibition of the channel activity ofASIC1a may reduce this harmful calcium flux, thereby reducingischemia-induced injury.

FIG. 2 shows a flowchart 30 with exemplary steps 32, 34 that may beperformed in a method of identifying drugs for treatment of ischemia.The steps may be performed any suitable number of times and in anysuitable combination. In the method, one or more ASIC-selectiveinhibitors may be obtained, indicated at 32. The inhibitors then may betested on an ischemic subject for an effect on ischemia-induced injury,indicated at 34.

Nerve Injuries

The present application is directed to pharmaceutical compositions andmethods for reducing nerve injuries in a subject. As used herein, theterm “nerve injury” means an acute or chronic injury to or adversecondition of a nervous system tissue or cell resulting from physicaltransaction or trauma, contusion or compression or surgical lesion,vascular pharmacologic insults including hemorrhagic or ischemic damage,or from neurodegenerative or any other neurological disease, or anyother factor causing the injury to or adverse condition of the nervoussystem tissue or cell. In some embodiments, the nerve injury is causedby cognitive disorders, psychotic disorders, neurotransmitter-mediateddisorders or neuronal disorders. Nerve injury includes injuries to thenervous system (i.e., nervous system injuries) and brain injury.

As used herein, the term “cognitive disorders” refers to and intendsdiseases and conditions that are believed to involve or be associatedwith or do involve or are associated with progressive loss of structureand/or function of neurons, including death of neurons, and where acentral feature of the disorder may be the impairment of cognition(e.g., memory, attention, perception and/or thinking). These disordersinclude pathogen-induced cognitive dysfunction, e.g. HIV associatedcognitive dysfunction or Lyme disease associated cognitive dysfunction.In some embodiments, the cognitive disorders are degenerative cognitivedisorders. Examples of degenerative cognitive disorders includeAlzheimer's Disease, Huntington's Disease, Parkinson's Disease,amyotrophic lateral sclerosis (ALS), autism, mild cognitive impairment(MCI), stroke, traumatic brain injury (TBI), age-associated memoryimpairment (AAMI) and epilepsy.

As used herein, the term “psychotic disorders” refers to and intendsmental diseases or conditions that are believed to cause or do causeabnormal thinking and perceptions. Psychotic disorders are characterizedby a loss of reality which may be accompanied by delusions,hallucinations (perceptions in a conscious and awake state in theabsence of external stimuli which have qualities of real perception, inthat they are vivid, substantial, and located in external objectivespace), personality changes and/or disorganized thinking. Other commonsymptoms include unusual or bizarre behavior, as well as difficulty withsocial interaction and impairment in carrying out the activities ofdaily living. Exemplary psychotic disorders are schizophrenia, bipolardisorders, psychosis, anxiety, depression and chronic pain.

As used herein, the term “neurotransmitter-mediated disorders” refers toand intends diseases or conditions that are believed to involve or beassociated with or do involve or are associated with abnormal levels ofneurotransmitters such as histamine, glutamate, serotonin, dopamine,norepinephrine or impaired function of aminergic G protein-coupledreceptors. Exemplary neurotransmitter-mediated disorders include spinalcord injury, diabetic neuropathy, allergic diseases and diseasesinvolving geroprotective activity such as age-associated hair loss(alopecia), age-associated weight loss and age-associated visiondisturbances (cataracts). Abnormal neurotransmitter levels areassociated with a wide variety of diseases and conditions including, butnot limited, to Alzheimer's disease, Parkinson's Disease, autism,Guillain-Barre syndrome, mild cognitive impairment, schizophrenia,anxiety, multiple sclerosis, stroke, traumatic brain injury, spinal cordinjury, diabetic neuropathy, fibromyalgia, bipolar disorders, psychosis,depression and a variety of allergic diseases.

As used herein, the term “neuronal disorders” refers to and intendsdiseases or conditions that are believed to involve, or be associatedwith, or do involve or are associated with neuronal cell death and/orimpaired neuronal function or decreased neuronal function. Exemplaryneuronal indications include neurodegenerative diseases and disorderssuch as Alzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis (ALS), Parkinson's disease, canine cognitive dysfunctionsyndrome (CCDS), Lewy body disease, Menkes disease, Wilson disease,Creutzfeldt-Jakob disease, Fahr disease, an acute or chronic disorderinvolving cerebral circulation, such as ischemic or hemorrhagic strokeor other cerebral hemorrhagic insult, age-associated memory impairment(AAMI), mild cognitive impairment (MCI), injury-related mild cognitiveimpairment (MCI), post-concussion syndrome, post-traumatic stressdisorder, adjuvant chemotherapy, traumatic brain injury (TBI), neuronaldeath mediated ocular disorder, macular degeneration, age-relatedmacular degeneration, autism, including autism spectrum disorder,Asperger syndrome, and Rett syndrome, an avulsion injury, a spinal cordinjury, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis,diabetic neuropathy, fibromyalgia, neuropathy associated with spinalcord injury, schizophrenia, bipolar disorder, psychosis, anxiety ordepression, and chronic pain..

In some embodiments, the nerve injuries or nervous system injuries arecaused by a change in the ion flux into neurons or a nervous systemtissue. As used herein, the term “nervous system tissue” refers toanimal tissue comprising nerve cells, the neuropil, glia, neuralinflammatory cells, and endothelial cells in contact with “ nervoussystem tissue”. “Nerve cells” may be any type of nerve cell known tothose of skill in the art including, but not limited to neurons. As usedherein, the term “neuron” represents a cell of ectodermal embryonicorigin derived from any part of the nervous system of an animal. Neuronsexpress well-characterized neuron-specific markers, includingneurofilament proteins, NeuN (Neuronal Nuclei marker), MAP2, and classIII tubulin. Included as neurons are, for example, hippocampal,cortical, midbrain dopaminergic, spinal motor, sensory, enteric,sympathetic, parasympathetic, septal cholinergic, central nervous systemand cerebellar neurons. “Glial cells” useful in the present inventioninclude, but are not limited to astrocytes, Schwan cells, andoligodendrocytes. “Neural inflammatory cells” useful in the presentinvention include, but are not limited to cells of myeloid originincluding macrophages and microglia.

In some embodiments, the pharmaceutical compositions and methods of thepresent application relate to reducing nerve injuries caused by ischemiaor an ischemia-related condition. Ischemia, as used herein, is a reducedblood flow to an organ(s) and/or tissue(s). The reduced blood flow maybe caused by many mechanisms, including but are not limited to, apartial or complete blockage (an obstruction), a narrowing (aconstriction), and/or a leak/rupture , of one or more blood vessels thatsupply blood to the organ(s) and/or tissue(s). Ischemia may be createdby thrombosis, an embolism, atherosclerosis, hypertension, hemorrhage,an aneurysm, surgery, trauma, medication, and the like. The reducedblood flow thus may be chronic, transient, acute or sporadic.

Any organ or tissue may experience a reduced blood flow and requiredtreatment for ischemia. Exemplary organs and/or tissues include, but arenot limited to, brain, arteries, heart, intestines and eye (e.g., theoptic nerve). Ischemia-induced injuries (i.e., disease and/or damageproduced by various types of ischemia) include, but are not limited to,ischemic myelopathy, ischemic optic neuropathy, ischemic colitis,coronary heart disease, and/or cardiac heart disease (e.g., angina,heart attack, etc.), among others. Ischemia-induced injury thus maydamage and/or kill cells and/or tissue, for example, producing necrotic(infarcted) tissue, inflammation, and/or tissue remodeling, amongothers, at affected sites within the body. Treatment according toaspects of the present application may reduce the incidence, extent,and/or severity of this injury.

An ischemia-related condition may be any consequence of ischemia. Theconsequence may be substantially concurrent with the onset ischemia(e.g., a direct effect of the ischemia) and/or may occur substantiallyafter ischemia onset and/or even after the ischemia is over (e.g., anindirect, downstream effect of the ischemia, such reperfusion of tissuewhen ischemia ends). Exemplary ischemia-related conditions may includeany combination of the symptoms (and/or conditions) listed above.Alternatively, or in addition, the symptoms may include local and/orsystemic acidosis (pH decrease), hypoxia (oxygen decrease), free radicalgeneration, and/or the like.

In some embodiments, the ischemia-related condition is stroke. Stroke,as used herein, is brain ischemia produced by a reduced blood supply toa part (or all) of the brain. Symptoms produced by stroke may be sudden(such as loss of consciousness) or may have a gradual onset over hoursor days. Furthermore, the stroke may be a major ischemic attack (a fullstroke) or a more minor, transient ischemic attack, among others.Symptoms produced by stroke may include, for example, hemiparesis,hemiplegia, one-sided numbness, one-sided weakness, one-sided paralysis,temporary limb weakness, limb tingling, confusion, trouble speaking,trouble understanding speech, trouble seeing in one or both eyes, dimvision, loss of vision, trouble walking, dizziness, a tendency to fall,loss of coordination, sudden severe headache, noisy breathing, and/orloss of consciousness. Alternatively, or in addition, the symptoms maybe detectable more readily or only via tests and/or instruments, forexample, an ischemia blood test (e.g., to test for altered albumin,particular protein isoforms, damaged proteins, etc.), anelectrocardiogram, an electroencephalogram, an exercise stress test,brain CT or MRI scanning and/or the like.

Acid-base balance is important for biological systems. Normal brainfunction depends on the complete oxidation of glucose, with the endproduct of CO₂ and H₂O for its energy requirements. During ischemia,increased anaerobic glycolysis, due to the lack of oxygen supply, leadsto lactic acid accumulation. Accumulation of lactic acid, along withincreased H⁺ release from ATP hydrolysis, causes decreases in tissue pH.Extracellular pH (pH_(o)) typically falls to 6.5 during ischemia, and itcan fall below 6.0 during severe ischemia or under hyperglycemicconditions.

Subjects of Nerve Injury

The method and pharmaceutical composition of the present application canbe used in any subject that has a nerve injury or a history of nerveinjury and/or a significant chance of developing nerve injury aftertreatment begins and during a time period in which the treatment isstill effective. In some embodiments, the subject is an ischemicsubjects. An ischemic subject, as used herein, is any person (a humansubject) or animal (an animal subject) that has ischemia, anischemia-related condition, a history of ischemia, and/or a significantchance of developing ischemia after treatment begins and during a timeperiod in which the treatment is still effective.

The subject may be an animal. The term “animal,” as used herein, refersto any animal that is not human. Exemplary animals that may be suitableinclude any animal with a bloodstream, such as rodents (mice, rats,etc.), dogs, cats, birds, sheep, goats, non-human primates, etc. Theanimal may be treated for its own sake, e.g., for veterinary purposes(such as treatment of a pet). Alternatively, the animal may provide ananimal model of nerve injury, such as ischemia, to facilitate testingdrug candidates for human use, such as to determine the candidates'potency, window of effectiveness, side effects, etc.

Ischemic subjects for treatment may be selected by any suitablecriteria. Exemplary criteria may include any detectable symptoms ofischemia, a history of ischemia, an event that increases the risk of (orinduces) ischemia (such as a surgical procedure, trauma, administrationof a medication, etc.), and/or the like. A history of ischemia mayinvolve one or more prior ischemic episodes. In some examples, a subjectselected for treatment may have had an onset of ischemia that occurredat least about one, two, or three hours before treatment begins, or aplurality of ischemic episodes (such as transient ischemic attacks) thatoccurred less than about one day, twelve hours, or six hours prior toinitiation of treatment.

Asic Inhibitors, Amiloride and Amiloride Analogs

Inhibitors of ASIC family members, as used herein, are substances thatreduce (partially, substantially, or completely block) the activity orone or more members of the ASIC family, that is, ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3, and ASIC4, among others. In some examples, the inhibitorsmay reduce the channel activity of one or more members, such as theability of the members to flux ions (e.g., sodium, calcium, and/orpotassium ions, among others) through cell membranes (into and/or out ofcells). The substances may be compounds (small molecules of less thanabout 10 kDa, peptides, nucleic acids, lipids, etc.), complexes of twoor more compounds, and/or mixtures, among others. Furthermore, thesubstances may inhibit ASIC family members by any suitable mechanismincluding competitive, noncompetitive, uncompetitive, and/or mixedinhibition, among others.

The inhibitor may be an ASIC1a inhibitor that inhibits acid sensing ionchannel 1a (ASIC1a). ASIC1a, as used herein, refers to an ASIC1a proteinor channel from any species. For example, an exemplary human ASIC1aprotein/channel is described in Waldmann, R., et al. 1997, Nature 386,pp. 173-177, which is incorporated herein by reference.

The expression “ASIC1a inhibitor” may refer to a product which, withinthe scope of sound pharmacological judgment, is potentially or actuallypharmaceutically useful as an inhibitor of ASIC1a, and includesreference to substances which comprise a pharmaceutically active speciesand are described, promoted, or authorized as an ASIC1a inhibitor.

An ASIC1a inhibitor may be selective within the ASIC family. Selectiveinhibition of ASIC1a, as used herein, is inhibition that issubstantially stronger on ASIC1a than on another ASIC family member(s)when compared (for example, in cultured cells) after exposure of each tothe same (sub-maximal) concentration(s) of an inhibitor. The inhibitormay inhibit ASIC1a selectively relative to at least one other ASICfamily member (ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC 4, etc.) and/orselectively relative to every other ASIC family member. The strength ofinhibition for a selective inhibitor may be described by an inhibitorconcentration at which inhibition occurs (e.g., an IC₅₀ (inhibitorconcentration that produces 50% of maximal inhibition) or a K₁ value(inhibition constant or dissociation constant)) relative to differentASIC family members. An ASIC1a-selective inhibitor may inhibit ASIC1aactivity at a concentration that is at least about two-, four-, orten-fold lower (one-half, one-fourth, or one-tenth the concentration orlower) than for inhibition of at least one other or of every other ASICfamily member. Accordingly, an ASIC1a-selective inhibitor may have anIC₅₀ and/or K_(i) for ASIC1a inhibition that is at least about two-,four-, or ten-fold lower (one-half, one-fourth, or one-tenth or less)than for inhibition of at least one other ASIC family member and/or forinhibition of every other ASIC family member.

An ASIC1a-selective inhibitor, in addition to being selective, also maybe specific for ASIC1a. ASIC1a-specific inhibition, as used herein, isinhibition that is substantially exclusive to ASIC1a relative to everyother ASIC family member, such as ASIC2a and ASIC3a. An ASIC1a-specificinhibitor may inhibit ASIC1a at an inhibitor concentration that is atleast about twenty-fold lower (5% of the concentration or less) than forinhibition of every other ASIC family member. Accordingly, anASIC1a-specific inhibitor may have an IC₅₀ and/or K_(i) for ASIC1arelative to every other member of the ASIC family that is at least abouttwenty-fold lower (five percent or less), such that, for example,inhibition of other ASIC family members is at least substantially (orcompletely) undetectable. In some embodiments, the ASIC1a-selectiveinhibitor has increased potency to homomeric ASIC1a channel andincreased aqueous solubility comparing to the commercially availableamiloride-related ASIC1a inhibitors such as amiloride benzamil,phenamil, 5-(N-ethyl-N-isobutyl) amiloride (EIPA), bepridil, KB-R7943,5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and5-(N,N-dimenthyl) amiloride hydrochloride.

Any suitable ASIC inhibitor or combination of inhibitors may be used.For example, a subject may be treated with an ASIC1a-selective inhibitorand a nonselective ASIC inhibitor, or with an ASIC1a-selective inhibitorand an inhibitor to a non-ASIC channel protein, such as a non-ASICcalcium channel. In some examples, a subject may be treated with anASIC1a-selective inhibitor and an inhibitor of NMDA receptors, such as aglutamate antagonist.

The inhibitor may be or include a peptide. The peptide may have anysuitable number of amino acid subunits, generally at least about ten andless than about one-thousand subunits. In some examples, the peptide mayhave a cystine knot motif. A cystine knot, as used herein, generallycomprises an arrangement of six or more cysteines. A peptide with thesecysteines may create a “knot” including (1) a ring formed by twodisulfide bonds and their connecting backbone segments, and (2) a thirddisulfide bond that threads through the ring. In some examples, thepeptide may be a conotoxin from an arachnid and/or cone snail species.For example, the peptide may be PcTx1 (psalmotoxin 1), a toxin from atarantula (Psalmopoeus cambridgei (Pc)).

In some examples, the peptide may be structurally related to PcTx1, suchthat the peptide and PcTx1 differ by at least one deletion, insertion,and/or substitution of one or more amino acids. For example, the peptidemay have at least about 25% or at least about 50% sequence identity,and/or at least about 25% or at least about 50% sequence similarity withPcTx1 (see below). Further aspects of peptides that may be suitable asinhibitors are described below in Example 3.

Methods of alignment of amino acid sequences for comparison andgeneration of identity and similarity scores are well known in the art.Exemplary alignment methods that may be suitable include (Best Fit) ofSmith and Waterman, a homology alignment algorithm (GAP) of Needlemanand Wunsch, a similarity method (Tfasta and Fasta) of Pearson andLipman, and/or the like. Computer algorithms of these and otherapproaches that may be suitable include, but are not limited to:CLUSTAL, GAP, BESTFIT, BLASTP, FASTA, and TFASTA.

As used herein, “sequence identity” or “identity” in the context of twopeptides relates to the percentage of residues in the correspondingpeptide sequences that are the same when aligned for maximumcorrespondence. In some examples, peptide residue positions that are notidentical may differ by conservative amino acid substitutions, whereamino acid residues are substituted for other amino acid residues withsimilar chemical properties (e.g. charge or hydrophobicity) andtherefore are expected to produce a smaller (or no) effect on thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards, to give a “similarity” of the sequences, whichcorrects for the conservative nature of the substitutions. For example,each conservative substitution may be scored as a partial rather than afull mismatch, thereby correcting the percentage sequence identity toprovide a similarity score. The scoring of conservative substitutions toobtain similarity scores is well known in the art and may be calculatedby any suitable approach, for example, according to the algorithm ofMeyers and Miller, Computer Applic. Biol Sci., 4: 11-17 (1988), e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

Amiloride, a guanidinium group containing pyrazine derivative, has beenused for the treatment of mild hypertension with little reported sideeffect. Amiloride works by directly blocking the epithelial sodiumchannel (ENaC) thereby inhibiting sodium reabsorption in the late distalconvoluted tubules, connecting tubules, and collecting ducts in thekidneys. This promotes the loss of sodium and water from the body, butwithout depleting potassium. As used herein, the term “amiloride” refersto both amiloride and salts of amiloride, such as amiloridehydrochloride.

Amiloride analogs, as used herein, refer to chemical compounds havingbiological activities similar to those of amiloride but with a slightlyaltered chemical structure. Examples of amiloride analogs include, butare not limited to, benzamil, phenamil, 5-(N-ethyl-N-isobutyl) amiloride(EIPA), bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride,5-(N,N-hexamethylene) amiloride and 5-(N,N-dimenthyl) amiloridehydrochloride. Other examples include amiloride analogs with ahydrophobic substitutent at the C₅-NH₂ position and/or on the guanidinogroup, as shown in FIG. 21, as well as methylated analogs of benzamil,amiloride analogs containing a ring formed on a guanidine group,amiloride analogs containing an acylguanidino group, and amilorideanalogs containing a water solubilizing group formed on a guanidinegroup, wherein the water solubilizing group is a N,N-dimethyl aminogroup or a sugar group. In some embodiments, the amiloride analogs donot to block the human Na⁺/Ca²⁺ ion exchanger. In other embodiments, theamiloride analogs are weak inhibitor of the Na⁺/Ca²⁺ ion exchanger andhelp maintaining low levels of intracellular Ca²⁺. In other embodiments,the amiloride analogs are very weak inhibitor of the Na⁺/Ca²⁺ ionexchanger with an IC₅₀ of 1.1 mM or less. In some other embodiments, theamiloride analogs do not block the ASIC2a and/or ASIC3 channels. In oneembodiment, the amiloride analogs have increased selectivity for ASIC1aover the ASIC3 channel and/or ASIC2 channel.

As used herein, the term “amiloride analog” refers to both amilorideanalog and salts of amiloride analog, such as 5-(N,N-dimenthyl)amiloridehydrochloride.

In some embodiments, amiloride and/or amiloride analogs are used inconjunction with other ASIC inhibitors such as PcTx1 and derivativesthereof

Administration of Inhibitors

Administration (or administering), as used herein, includes any route ofsubject exposure to an inhibitor, under any suitable conditions, and atany suitable time(s). Administration may be self-administration oradministration by another, such as a health-care practitioner (e.g., adoctor, a nurse, etc.). Administration may be by injection (e.g.,intravenous, intramuscular, subcutaneous, intracerebral,introcerebroventricular, epidural, and/or intrathecal, among others),ingestion (e.g., using a capsule, lozenge, a fluid composition, etc.),inhalation (e.g., an aerosol (less than about 10 microns average dropletdiameter) inhaled nasally and/or orally), absorption through the skin(e.g., with a skin patch) and/or mucosally (e.g., through oral, nasal,and/or pulmonary mucosa, among others), and/or the like. Mucosaladministration may be achieved, for example, using a spray (such as anasal spray), an aerosol that is inhaled), and/or the like. A spray maybe a surface spray (droplets on average greater than about 50 microns indiameter) and/or a space spray (droplets on average about 10-50 micronsin diameter). In some examples, ischemia may produce an alteration ofthe blood-brain barrier of an ischemic subject, thus increasing theefficiency with which an inhibitor that is introduced (e.g., byinjection and/or absorption) into the bloodstream of a subject can reachthe brain. Administration may be performed once or a plurality of times,and at any suitable time relative to ischemia diagnosis, to providetreatment. Accordingly, administration may be performed before ischemiahas been detected (e.g., prophylactically,) after a minor ischemicepisode, during chronic ischemia, after a full stroke, and/or the like.In some embodiments, amiloride or amiloride analog is administeredintravenously. In other embodiments, amiloride or amiloride analog isadministered intracerebrally. In other embodiments, amiloride oramiloride analog is administered intracerebroventricularly. In otherembodiments, amiloride or amiloride analog is administeredintramuscularly. In other embodiments, amiloride or amiloride analog isadministered intrathecally.

A therapeutically effective amount (or simply “an effective amount”) ofan inhibitor may be administered. A therapeutically effective amount oran effective amount of an inhibitor, as used herein, is any amount ofthe inhibitor that, when administered to subjects, reduces, in asignificant number of the subjects, the degree, incidence, and/or extentof ischemia-induced injury in the subjects. Accordingly, atherapeutically effective amount may be determined, for example, inclinical studies in which various amounts of the inhibitor areadministered to test subjects (and, generally, compared to a controlgroup of subjects). The therapeutically effective amount of inhibitor orinhibitors may be given by a single injection or multiple injections ina volume of 0.1-50 ml per injection.

In some embodiments, the inhibitor is amiloride, an amiloride analog ora salt thereof and is given at a daily dose (as a single dose ormultiple dose) in the range of 0.01-30 mg/kg body weight, 0.01-10 mg/kgbody weight, 0.01-3 mg/kg body weight, 0.01-1 mg/kg body weight,0.01-0.3 mg/kg body weight, 0.01-0.1 mg/kg body weight, 0.01-0.03 mg/kgbody weight, 0.03-30 mg/kg body weight, 0.03-10 mg/kg body weight,0.03-3 mg/kg body weight, 0.03-1 mg/kg body weight, 0.03-0.3 mg/kg bodyweight, 0.03-0.1 mg/kg body weight, 0.1-30 mg/kg body weight, 0.1-10mg/kg body weight, 0.1-3 mg/kg body weight, 0.1-1 mg/kg body weight,0.1-0.3 mg/kg body weight, 0.3-30 mg/kg body weight, 0.3-10 mg/kg bodyweight, 0.3-3 mg/kg body weight, 0.3-1 mg/kg body weight, 1-30 mg/kgbody weight, 1-10 mg/kg body weight, 1-3 mg/kg body weight, 3-30 mg/kgbody weight, 3-10 mg/kg body weight or 10-30 mg/kg body weight. In oneembodiment, the amiloride analog is selected from the group consistingof benzamil, phenamil, EIPA, bepridil, KB-R7943,5-(N-methyl-N-isobutyl)-amiloride, 5-(N,N-hexamethylene)-amiloride and5-(N,N-dimenthyl)amiloride hydrochloride. In another embodiment, theamiloride analog has a hydrophobic substitutent at the C₅-NH₂ positionand/or on the guanidino group. In another embodiment, the amilorideanalog is selected from the amiloride analogs selected from the groupconsisting of methylated analogs of benzamil, amiloride analogscontaining a ring formed on a guanidine group, amiloride analogscontaining an acylguanidino group, and amiloride analogs containing awater solubilizing group formed on a guanidine group, wherein the watersolubilizing group is a N,N-dimethyl amino group or a sugar group.

In other embodiments, the inhibitor is amiloride, an amiloride analog ora salt thereof and is administered as a pharmaceutical compositionformulated as a single dose in the range of 0.1-1000 mg/dose, 0.1-300mg/dose, 0.1-100 mg/dose, 0.1-30 mg/dose, 0.1-10 mg/dose, 0.1-3 mg/dose,0.1-1 mg/dose, 0.1-0.3 mg/dose, 0.3-1000 mg/dose, 0.3-300 mg/dose,0.3-100 mg/dose, 0.3-30 mg/dose, 0.3-10 mg/dose, 0.3-3 mg/dose, 0.3-1mg/dose, 1-1000 mg/dose, 1-300 mg/dose, 1-100 mg/dose, 1-30 mg/dose,1-10 mg/dose, 1-3 mg/dose, 3-1000 mg/dose, 3-300 mg/dose, 3-100 mg/dose,3-30 mg/dose, 3-10 mg/dose, 10-1000 mg/dose, 10-300 mg/dose, 10-100mg/dose, 10-30 mg/dose, 30-1000 mg/dose, 30-300 mg/dose, 30-100 mg/dose,100-1000 mg/dose, 100-300 mg/dose, or 300-1000 mg/dose. In oneembodiment, the amiloride analog is selected from the group consistingof benzamil, phenamil, EIPA, bepridil, KB-R7943,5-(N-methyl-N-isobutyl)-amiloride, 5-(N,N-hexamethylene)-amiloride and5-(N,N-dimenthyl)amiloride hydrochloride. In another embodiment, theamiloride analog has a hydrophobic substitutent at the C₅-NH₂ positionand/or on the guanidino group. In another embodiment, the amilorideanalog is selected from the amiloride analogs selected from the groupconsisting of methylated analogs of benzamil, amiloride analogscontaining a ring formed on a guanidine group, amiloride analogscontaining an acylguanidino group, and amiloride analogs containing awater solubilizing group formed on a guanidine group, wherein the watersolubilizing group is a N,N-dimethyl amino group or a sugar group. Insome embodiments, the pharmaceutical composition formulated forintravenous injection, intracerebral injection, intracerebroventricularinjection, intrathecal injection or intramuscular injection.

The inhibitor may be administered in any suitable form and in anysuitable composition to subjects. In some examples, the inhibitor may beconfigured as a pharmaceutically acceptable salt. The composition may beformulated to include, for example, a fluid carrier/solvent (a vehicle),a preservative, one or more excipients, a coloring agent, a flavoringagent, a salt(s), an anti-foaming agent, and/or the like. The inhibitormay be present at a concentration in the vehicle that provides atherapeutically effective amount of the inhibitor for treatment ofischemia when administered to an ischemic subject.

In some embodiments, amiloride analogs with higher water solubility orlipid solubility are produced. In certain embodiments, the amilorideanalogs contain a water solubilizing group, such as an N,N-dimethylamino group or a sugar, at the guanidino group to improve watersolubility (formula 13-16, FIG. 24). In some embodiments, the amilorideanalogs have a water solubility of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or higher. In other embodiments,the amiloride analogs have a solubility that allows for a 10 mg, 25 mg,50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg dose tobe administered intravenously to a human in a single 10 ml injection. Inyet other embodiments, the amiloride analogs have a solubility thatallows for a 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300mg, 400 mg, or 500 mg dose to be administered intracerebroventicularlyto a human in a single 2 ml injection.

Synthesis and Screening of Amiloride Analogs

Another aspect of the present application relates to the synthesis andscreening of new amiloride analogs. Synthesis pathway of amilorideanalogs are designed based on the desired analog structure. The newlysynthesized amiloride analogs are then screened for their inhibitiveeffect on ASIC family members, such as ASIC1a and ASIC2a. One or moreASIC inhibitors, particularly ASIC1a inhibitors as described above, maybe obtained. The inhibitors may be obtained by any suitable approach,such by screening a set of candidate inhibitors (e.g., a library of twoor more compounds) and/or by rationale design, among others.

Screening may involve any suitable assay system that measuresinteraction between ASIC proteins and the set of candidate inhibitors.Exemplary assay systems may include assays performed biochemically(e.g., binding assays), with cells grown in culture (“cultured cells”),and/or with organisms, among others.

In some embodiments, a cell-based assay system is used to measure theeffect of each candidate inhibitor on ion flux, such as acid-sensitiveion flux, in the cells. In some embodiments, the ion flux is a flux ofcalcium and/or sodium. In some embodiments, the assay system uses cellsexpressing an ASIC family member, such as ASIC1a or ASIC2a, or two ormore distinct sets of cells expressing two or more distinct ASIC familymembers, such as ASIC1a and another ASIC family member(s), to determinethe selectivity of each inhibitor for these family members. The cellsmay express each ASIC family member endogenously or through introductionof foreign nucleic acid. In some examples, the assay system may measureion flux electrophysiologically (such as by patch clamp), using anion-sensitive or membrane potential-sensitive dye (e.g., a calciumsensitive dye such as Fura-2), or via a gene-based reporter system thatis sensitive to changes in membrane potential and/or intracellular ion(e.g., calcium) concentrations, among others. The assay system may beused to test candidate inhibitors for selective and/or specificinhibition of ASIC family members, particularly ASIC1a.

One or more ASIC inhibitors may be administered to a subject with anerve injury, such as an ischemic subject to test the efficacy of theinhibitors for treatment of the nerve injury. The ischemic subjects maybe people or animals. In some examples, the ischemic subjects mayprovide an animal model system of ischemia and/or stroke. Exemplaryanimal model systems include rodents (mice and/or rats, among others)with ischemia induced experimentally. The ischemia may be inducedmechanically (e.g., surgically) and/or by administration of a drug,among others. In some examples, the ischemia may be induced by occlusionof a blood vessel, such as by constriction of a mid-cerebral artery.

Another aspect of the present application relates to a pharmaceuticalcomposition for reducing nervous system injury. The pharmaceuticalcomposition comprises an effective amount of amiloride, an amilorideanalog or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated for intravenous, intrathecal orintracerebroventricular injection.

In some embodiments, the pharmaceutical composition comprises anamiloride analog or a pharmaceutically acceptable salt thereof, whereinthe amiloride analog is selected from the group consisting of benzamil,phenmil, EIPA bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride,5-(N,N-hexamethylene) amiloride and 5-(N,N-dimenthyl) amiloridehydrochloride.

In other embodiments, the pharmaceutical composition comprises anamiloride analog or a pharmaceutically acceptable salt thereof, whereinthe amiloride analog is selected from the group consisting of methylatedanalogs of benzamil, amiloride analogs containing a ring formed on aguanidine group, amiloride analogs containing an acylguanidino group,and amiloride analogs containing a water solubilizing group formed on aguanidine group, wherein the water solubilizing group is a N,N-dimethylamino group or a sugar group.

In other embodiments, the pharmaceutical composition further comprisesone or more other ASIC inhibitors. In one embodiment, the one or moreother ASIC inhibitors comprise PcTx1 or a PcTx1 derivative.

Another aspect of the present application relates to a pharmaceuticalcomposition for reducing nervous system injury. The pharmaceuticalcomposition comprises an effective amount of an amiloride analog or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.

In some embodiments, the pharmaceutical composition is formulated forintravenous, intrathecal, intracerebroventricular or intramuscularinjection.

In one embodiment, the amiloride analog is a methylated analog ofbenzamil. In another embodiment, the amiloride analog comprises a ringformed on a guanidine group. In another embodiment, the amiloride analogcomprises an acylguanidino group. In yet another embodiment, theamiloride analog comprises a water solubilizing group formed on aguanidine group, wherein the water solubilizing group is a N,N-dimethylamino group or a sugar group.

In other embodiments, the pharmaceutical composition further comprisesone or more other ASIC inhibitors. In one embodiment, the one or moreother ASIC inhibitors comprise PcTx1 or a PcTx1 derivative.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, sweeteners and thelike. The pharmaceutically acceptable carriers may be prepared from awide range of materials including, but not limited to, flavoring agents,sweetening agents and miscellaneous materials such as buffers andabsorbents that may be needed in order to prepare a particulartherapeutic composition. The use of such media and agents withpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Optionally, the amiloride and/or an amiloride analog maybe mixed together into pharmaceutical compositions comprisingsupplementary active ingredients that are not contraindicated by saidamiloride and/or an amiloride analog.

In some embodiments, the pharmaceutical composition is formulated forintravenous injection. In other embodiments, the pharmaceuticalcomposition comprises amiloride and/or amiloride analog formulated forintravenous injection. In other embodiments, the pharmaceuticalcomposition comprises amiloride and/or amiloride analog formulated forintracerebroventricular injection. In other embodiments, thepharmaceutical composition comprises amiloride and/or amiloride analogformulated for intrathecal injection. In other embodiments, thepharmaceutical composition comprises amiloride and/or amiloride analogformulated for intramuscular injection. Pharmaceutical compositionssuitable for injectable use include sterile aqueous solutions (wherewater soluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the injectablecompositions are sterile and are fluid to the extent that easysyringability exists. The injectable composition must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating amilorideand/or amiloride analog in the required amount in an appropriatesolvent, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active, ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof

In some embodiments, the pharmaceutical composition is provided in a dryform, and is formulated into a tablet or capsule form. Tablets may beformulated in accordance with conventional procedures employing solidcarriers well-known in the art. Hard and soft capsules employed in thepresent invention can be made from any pharmaceutically acceptablematerial, such as gelatin or cellulosic derivatives.

In certain embodiments, the pharmaceutical composition is formulated forimmediate release, extended-release, delayed-release or combinationsthereof. Extended-release, also known as sustained-release, time-releaseor timed-release, controlled-release (CR), modified release (MR), orcontinuous-release (CR or Contin), is a mechanism used in medicinetablets or capsules to dissolve slowly and release the active ingredientover time. The advantages of sustained-release tablets or capsules arethat they can often be taken less frequently than instant-releaseformulations of the same drug, and that they keep steadier levels of thedrug in the bloodstream, thus extending the duration of the drug action.

In one embodiment, the pharmaceutical composition is formulated forextended release by embedding the active ingredient in a matrix ofinsoluble substance(s) such as acrylics or chitin. A extended releaseform is designed to release the active ingredient at a predeterminedrate by maintaining a constant drug level for a specific period of time.This can be achieved through a variety of formulations, including, butnot limited to liposomes and drug-polymer conjugates, such as hydrogels.

In another embodiment, the pharmaceutical composition is formulated fordelayed-release, such that the active ingredient(s) is not immediatelyreleased upon administration. A non-limiting example of a delayedrelease vehicle is an enteric coated oral medication that dissolves inthe intestines rather than the stomach.

In other embodiments, the pharmaceutical composition is formulated forimmediate release of a portion of the active ingredient, followed withan extended-release of the remainder of the active ingredient. In oneembodiment, the pharmaceutical composition is formulated as a powderthat can be ingested for rapid release of the active ingredient. Inanother embodiment, the pharmaceutical composition is formulated into aliquid, gel, liquid suspension or emulsion form. Said liquid, gel,suspension or emulsion may be ingested by the subject in naked form orcontained within a capsule.

In yet another embodiment, the pharmaceutical composition may beprovided as a skin or transdermal patch for the topical administrationof controlled and/or sustained quantities of the active ingredient.

EXAMPLES

The following examples describes selected aspects and embodiments of thepresent teachings, particularly data describing in vitro and in vivoeffects of ASIC inhibition, and exemplary cystine knot peptides for useas inhibitors. These examples are intended for the purposes ofillustration and should not be construed to limit the scope of thepresent teachings.

Example 1: Neuroprotection in Ischemia by Blocking Calcium-PermeableAcid-Sensing Ion Channels

This example describes experiments showing a role of ASIC1a in mediatingischemic injury and the ability ASIC1a inhibitors to reduce ischemicinjury; see FIGS. 2-10. Ca²⁺ toxicity may play a central role inischemic brain injury. The mechanism by which toxic Ca²⁺ loading ofcells occurs in the ischemic brain has become less clear as multiplehuman trials of glutamate antagonists have failed to show effectiveneuroprotection in stroke. Acidosis is a common feature of ischemia andplays a critical role in brain injury. This example demonstrates thatacidosis activates Ca²⁺-permeable acid-sensing ion channels (ASICs),which may induce glutamate receptor-independent, Ca²⁺-dependent,neuronal injury. Accordingly, cells lacking endogenous ASICs may beresistant to acid injury, while transfection of Ca²⁺-permeable ASIC1amay establish sensitivity. In focal ischemia, intracerebroventricularinjection of ASIC1a blockers or knockout of the ASIC1a gene may protectthe brain from ischemic injury and may do so more potently thanglutamate antagonism.

The normal brain requires complete oxidation of glucose to fulfill itsenergy requirements. During ischemia, oxygen depletion forces the brainto switch to anaerobic glycolysis. Accumulation of lactic acid as abyproduct of glycolysis and protons produced by ATP hydrolysis causes pHto fall in the ischemic brain and aggravates ischemic brain injury.

Acid-sensing ion channels (ASICs) are a class of ligand-gated channelsexpressed throughout neurons of mammalian central and peripheral nervoussystems. To date, six ASIC subunits have been cloned. Four of thesesubunits form functional homomultimeric channels that are activated byacidic pH to conduct a sodium-selective, amiloride-sensitive, cationcurrent. Two of the ASIC subunits, ASIC1a and ASIC2a subunits, have beenshown to be abundant in the brain.

Experimental Procedures Neuronal Culture

Following anesthesia with halothane, cerebral cortices were dissectedfrom E16 Swiss mice or P1 ASIC1^(+/+) and ASIC1^(−/−) mice and incubatedwith 0.05% trypsin-EDTA for 10 min at 37° C. Tissues were thentriturated with fire-polished glass pipettes and plated onpoly-L-ornithine-coated 24-well plates or 25×25 mm glass coverslips at adensity of 2.5×10⁵ cells per well or 10⁶ cells per coverslip. Neuronswere cultured with MEM supplemented with 10% horse serum (for E16cultures) or Neurobasal medium supplemented with B27 (for P1 cultures)and used for electrophysiology and toxicity studies after 12 days. Glialgrowth was suppressed by addition of 5-fluoro-2-deoxyuridine anduridine, yielding cultured cells with 90% neurons as determined by NeuNand GFAP staining (data not shown).

Electrophysiology

ASIC currents were recorded with whole-cell patch-clamp andfast-perfusion techniques. The normal extracellular solution (ECF)contained (in mM) 140 NaCl, 5.4 KCl, 25 HEPES, 20 glucose, 1.3 CaCl₂,1.0 MgCl₂, 0.0005 TTX (pH 7.4), 320-335 mOsm. For low pH solutions,various amounts of HCl were added. For solutions with pH<6.0, MESinstead of HEPES was used for more reliable pH buffering. Patchelectrodes contained (in mM) 140 CsF, 2.0 MgCl₂, 1.0 CaC₂, 10 HEPES, 11EGTA, 4 MgATP (pH 7.3), 300 mOsm. The Na⁺-free solution consisted of 10mM CaCl₂, 25 mM HEPES with equiosmotic NMDG or sucrose substituting forNaCl (Chu et al., 2002). A multibarrel perfusion system (SF-77B, WarnerInstrument Co.) was employed for rapid exchange of solutions.

Cell Injury Assay—LDH Measurement

Cells were washed three times with ECF and randomly divided intotreatment groups. MK801 (10 μM), CNQX (20 μM), and nimodipine (5 μM)were added in all groups to eliminate potential secondary activation ofglutamate receptors and voltage-gated Ca²⁺ channels. Following acidincubation, neurons were washed and incubated in Neurobasal medium at37° C. LDH release was measured in culture medium using the LDH assaykit (Roche Molecular Biochemicals). Medium (100 μL) was transferred fromculture wells to 96-well plates and mixed with 100 μL reaction solutionprovided by the kit. Optical density was measured at 492 nm 30 minlater, utilizing a microplate reader (Spectra Max Plus, MolecularDevices). Background absorbance at 620 was subtracted. The maximalreleasable LDH was obtained in each well by 15 min incubation with 1%Triton X-100 at the end of each experiment.

Ca²⁺ Imaging

Cortical neurons grown on 25×25 mm glass coverslips were washed threetimes with ECF and incubated with 5 μM fura-2-acetoxymethyl ester for 40min at 22° C., washed three times, and incubated in normal ECF for 30min. Coverslips were transferred to a perfusion chamber on an invertedmicroscope (Nikon TE300). Cells were illuminated using a xenon lamp andobserved with a 40x UV fluor oil-immersion objective lens, and videoimages were obtained using a cooled CCD camera (Sensys KAF 1401,Photometrics). Digitized images were acquired and analyzed in a PCcontrolled by Axon Imaging Workbench software (Axon Instruments). Theshutter and filter wheel (Lambda 10-2) were controlled by the softwareto allow timed illumination of cells at 340 or 380 nm excitationwavelengths. Fura-2 fluorescence was detected at emission wavelength of510 nm. Ratio images (340/380) were analyzed by averaging pixel ratiovalues in circumscribed regions of cells in the field of view. Thevalues were exported to SigmaPlot for further analysis.

Fluorescein-Diacetate Staining and Propidium Iodide Uptake

Cells were incubated in ECF containing fluorescein-diacetate (FDA) (5μM) and propidium iodide (PI) (2 μM) for 30 min followed by wash withdye-free ECF. Alive (FDA-positive) and dead (PI-positive) cells wereviewed and counted on a microscope (Zeiss) equipped with epifluorescenceat 580/630 nm excitation/emission for PI and 500/550 nm for FDA. Imageswere collected using an Optronics DEI-730 camera equipped with a BQ 8000sVGA frame grabber and analyzed using computer software (Bioquant,Tenn.).

Transfection of COS-7 Cells

COS-7 cells were cultured in MEM with 10% HS and 1% PenStrep (GIBCO). At˜50% confluence, cells were cotransfected with cDNAs for ASICs and GFPin pc^(DNA3) vector using FuGENE6 transfection reagents (Roche MolecularBiochemicals). DNA for ASICs (0.75 μg) and 0.25 μg of DNA for GFP wereused for each 35 mm dish. GFP-positive cells were selected forpatch-clamp recording 48 hr after transfection. For stable transfectionof ASIC1a, 500 m/mL G418 was added to culture medium I week followingthe transfection. The surviving G418-resistant cells were further platedand passed for >5 passages in the presence of G418. Cells were thenchecked with patch-clamp and immunofluorescent staining for theexpression of ASIC1a.

Oxygen-Glucose Deprivation

Neurons were washed three times and incubated with glucose-free ECF atpH 7.4 or 6.0 in an anaerobic chamber (Model 1025, Forma Scientific)with an atmosphere of 85% N₂, 10% H₂, and 5% CO₂ at 35° C.Oxygen-glucose deprivation (OGD) was terminated after 1 hr by replacingthe glucose-free ECF with Neurobasal medium and incubating the culturesin a normal cell culture incubator. With HEPES-buffered ECF used, 1 hrOGD slightly reduced pH from 7.38 to 7.28 (n=3) and from 6.0 to 5.96(n=4). Focal Ischemia

Transient focal ischemia was induced by suture occlusion of the middlecerebral artery (MCAO) in male rats (SD, 250-300 g) and mice (withcongenic C57B16 background, ˜25 g) anesthetized using 1.5% isoflurane,70% N₂O, and 28.5% O₂ with intubation and ventilation. Rectal andtemporalis muscle temperature was maintained at 37° C.±0.5° C. with athermostatically controlled heating pad and lamp. Cerebral blood flowwas monitored by transcranical LASER doppler. Animals with blood flownot reduced below 20% were excluded.

Animals were killed with chloral hydrate 24 hr after ischemia. Brainswere rapidly removed, sectioned coronally at 1 mm (mice) or 2 mm (rats)intervals, and stained by immersion in vital dye (2%)2,3,5-triphenyltetrazolium hydrochloride (TTC). Infarction area wascalculated by subtracting the normal area stained with TTC in theischemic hemisphere from the area of the nonischemic hemisphere. Infarctvolume was calculated by summing infarction areas of all sections andmultiplying by slice thickness. Rat intraventricular injection wasperformed by stereotaxic technique using a microsyringe pump withcannula inserted stereotactically at 0.8 mm posterior to bregma, 1.5 mmlateral to midline, and 3.8 mm ventral to the dura. All manipulationsand analyses were performed by individuals blinded to treatment groups.

Results (a) Acidosis Activates ASICs in Mouse Cortical Neurons

FIGS. 3A-D and 4A-D shows exemplary data related to theelectrophysiology and pharmacology of ASICs in cultured mouse corticalneurons. FIGS. 3A and 3B are graphs illustrating the pH dependence ofASIC currents activated by a pH drop from 7.4 to the pH valuesindicated. Dose-response curves were fit to the Hill equation with anaverage pH_(0.5) of 6.18±0.06 (n=10). FIGS. 3C and 3D are graphsillustrating the current-voltage relationship of ASICs (n=5). Theamplitudes of ASIC current at various voltages were normalized to thatrecorded at −60 mV. FIGS. 4A and 4B are graphs illustrating adose-dependent blockade of ASIC currents by amiloride. IC₅₀=16.4±4.1 μM,N=8. FIGS. 4C and 4D are graphs illustrating a blockade of ASIC currentsby PcTX venom. **p<0.01.

ASIC currents in cultured mouse cortical neurons were recorded (see FIG.3). At a holding potential of −60 mV, a rapid reduction of extracellularpH (pH,) to below 7.0 evoked large transient inward currents with asmall steady-state component in the majority of neurons (FIG. 3A). Theamplitude of inward current increased in a sigmoidal fashion as pH,decreased, yielding a pH_(0.5) of 6.18±0.06 (n=10, FIG. 3B). A linearI-V relationship and a reversal close to the Na⁺ equilibrium potentialwere obtained (n=6, FIGS. 3C and 3D). These data demonstrate thatlowering pH, may activate typical ASICs in mouse cortical neurons.

The effect of amiloride, a nonspecific blocker of ASICs, on theacid-activated currents was tested (see FIG. 4). As shown in FIGS. 4A-D,amiloride dose-dependently blocked ASIC currents in cortical neuronswith an IC₅₀ of 16.4±4.1 μM (n=8, FIGS. 4A and 4B). The effect of PcTXvenom on acid-activated current in cortical neurons is shown in FIG. 4Cand 4D. At 100 ng/mL, PcTX venom reversibly blocked the peak amplitudeof ASIC current by 47%±7% (n=15, FIGS. 4C and 4D), indicatingsignificant contributions of homomeric ASIC1a to total acid-activatedcurrents. Increasing PcTX concentration did not induce further reductionin the amplitude of ASIC current in the majority of cortical neurons(n=8, data not shown), indicating coexistence of PcTX-insensitive ASICs(e.g., heteromeric ASIC1a/2a) in these neurons.

(b) ASIC Response is Potentiated by Modeled Ischemia

FIGS. 5A-D show exemplary data indicating that modeled ischemia mayenhance activity of ASICs. FIG. 5A is a series of exemplary tracesshowing an increase in amplitude and a decrease in desensitization ofASIC currents following 1 hr OGD. FIG. 5B is a graph of summary dataillustrating an increase of ASIC current amplitude in OGD neurons. N=40and 44, *p<0.05. FIG. 5C is a series of exemplary traces and summarydata showing decreased ASIC current desensitization in OGD neurons. N=6,**p<0.01. FIG. 5D is a pair of exemplary traces showing lack ofacid-activated current at pH 6.0 in ASIC1^(−/−) neurons, in controlcondition, and following 1 hr OGD (n=12 and 13).

Since acidosis may be a central feature of brain ischemia, it wasdetermined to test whether ASICs may be activated in ischemic conditionsand whether ischemia may modify the properties of these channels; seeFIGS. 5A-D. ASIC currents in neurons following 1 hr oxygen glucosedeprivation (OGD) were recorded. Briefly, one set of cultures was washedthree times with glucose-free extracellular fluid (ECF) and subjected toOGD, while control cultures were subjected to washes with glucosecontaining ECF and incubation in a conventional cell culture incubator.OGD was terminated after 1 hr by replacing glucose-free ECF withNeurobasal medium and incubating cultures in the conventional incubator.ASIC current was then recorded 1 hr following the OGD when there was nomorphological alteration of neurons. OGD treatment induced a moderateincrease of the amplitude of ASIC currents (1520±138 pA in controlgroup, N=44; 1886±185 pA in neurons following 1 hr OGD, N=40, p<0.05,FIGS. 5A and 5B). More importantly, OGD induced a dramatic decrease inASIC desensitization as demonstrated by an increase in time constant ofthe current decay (814.7±58.9 ms in control neurons, N=6; 1928.9±315.7ms in neurons following OGD, N=6, p<0.01, FIGS. 5A and 5C). In corticalneurons cultured from ASIC1^(−/−) mice, reduction of pH from 7.4 to 6.0did not activate any inward current (n=52), similar to a previous studyin hippocampal neurons (Wemmie et al., 2002). In these neurons, 1 hr OGDdid not activate or potentiate acid-induced responses (FIG. 5D, n=12 and13).

(c) Acidosis Induces Glutamate-Independent Ca²⁺ Entry via ASIC1a

FIGS. 6A-B and 7A-D show exemplary data suggesting that ASICs incortical neurons may be Ca²⁺ permeable, and that Ca²⁺ permeability maybe ASIC1a dependent. FIG. 6A shows exemplary traces obtained withNa⁺-free ECF containing 10 mM Ca²⁺ as the only charge carrier. Inwardcurrents were recorded at pH 6.0. The average reversal potential is ˜−17mV after correction of liquid junction potential (n=5). FIG. 6B showsrepresentative traces and summary data illustrating blockade ofCa²-mediated current by amiloride and PcTX venom. The peak amplitude ofCa²-mediated current decreased to 26%±2% of control value by 100 μMamiloride (n=6, p<0.01) and to 22%±0.9% by 100 ng/mL PcTX venom (n=5,p<0.01). FIG. 7A shows exemplary 340/380 nm ratios as a function of pH,illustrating an increase of [Ca²⁺]_(i) by pH drop to 6.0. Neurons werebathed in normal ECF containing 1.3 mM CaCl₂ with blockers forvoltage-gated Ca²⁺ channels (5 μM nimodipine and 1μM w-conotoxin MVIIC)and glutamate receptors (10 μM MK801 and 20 μM CNQX). The inset of FIG.7A shows exemplary inhibition of acid-induced increase of [Ca²⁺]_(i) by100 μM amiloride. FIG. 7B shows exemplary summary data illustratinginhibition of acid-induced increase of [Ca²⁺]_(i) by amiloride and PcTXvenom. N=6-8, **p<0.01 compared with pH 6.0 group. FIG. 7C showsexemplary 340/380 nm ratios as a function of pH and NMDApresence/absence, demonstrating a lack of acid-induced increase of[Ca²⁺]_(i) in ASIC1^(−/−) neurons; neurons had a normal response to NMDA(n=8). FIG. 7D shows exemplary traces illustrating a lack ofacid-activated current at pH 6.0 in ASIC1^(−/−) neurons.

The Ca²⁺ permeability of ASICs in cortical neurons was determined usinga standard ion-substitution protocol (Jia et al., Neuron, 1996,17:945-956) and the Fura-2 fluorescent Ca²⁺-imaging technique (Chu etal., 2002, J. Neurophysiol. 87:2555-2561). With bath solutionscontaining 10 mM Ca²⁺ (Na⁺ and K⁺-free) as the only charge carrier andat a holding potential of -60 mV, we recorded inward currents largerthan 50 pA in 15 out of 18 neurons, indicating significant Ca²⁺permeability of ASICs in the majority of cortical neurons (FIG. 6A).Consistent with activation of homomeric ASIC1a channels, currentscarried by 10 mM Ca²⁺ were largely blocked by both the nonspecific ASICblocker amiloride and the ASIC1a-specific blocker PcTX venom (FIG. 6B).The peak amplitude of Ca²⁺-mediated current was decreased to 26%±2% ofcontrol by 100 μM amiloride (n=6, p<0.01) and to 22%±0.9% by 100 ng/mLPcTX venom (n=5, p<0.01). Ca²⁺ imaging, in the presence of blockers ofother major Ca²⁺ entry pathways (MK801 10 μM and CNQX 20 μM forglutamate receptors; nimodipine 5 μM and w-conotoxin MVIIC 1 μM forvoltage-gated Ca²⁺ channels), demonstrated that 18 out of 20 neuronsresponded to a pH drop with detectable increases in the concentration ofintracellular Ca²⁺ ([Ca²⁺]_(i) ) (FIG. 7A). In general, [Ca²⁺]_(i)remains elevated during prolonged perfusion of low pH solutions. In somecells, the [Ca²⁺]_(i) increase lasted even longer than the duration ofacid perfusion (FIG. 7A). Long-lasting Ca²⁺ responses suggest that ASICresponse in intact neurons may be less desensitized than in whole-cellrecordings or that Ca²⁺ entry through ASICs may induce subsequent Ca²⁺release from intracellular stores. Preincubation of neurons with 1μMthapsigargin partially inhibited the sustained component of Ca²⁺increase, suggesting that Ca²⁺ release from intracellular stores mayalso contribute to acid-induced intracellular Ca²⁺ accumulation (n=6,data not shown). Similar to the current carried by Ca²⁺ ions (FIG. 6B),both peak and sustained increases in [Ca²⁺]_(i) were largely inhibitedby amiloride and PcTX venom (FIGS. 7A and 7B, n=6-8), consistent withinvolvement of homomeric ASIC1a in acid-induced [Ca²⁺]_(i) increase.Knockout of the ASICI gene eliminated the acid-induced [Ca²⁺]_(i)increase in all neurons without affecting NMDA receptor-mediated Ca²⁺response (FIG. 7C, n=8). Patch-clamp recordings demonstrated lack ofacid-activated currents at pH 6.0 in 52 out of 52 of the ASIC1^(−/−)neurons, consistent with absence of ASIC1a subunits. Lowering pH to 5.0or 4.0, however, activated detectable current in 24 out of 52ASIC1^(−/−) neurons, indicating the presence of ASIC2a subunits in theseneurons (FIG. 7D). Further electrophysiological studies demonstratedthat ASIC1^(−/−) neurons have normal responses for various voltage-gatedchannels and NMDA, GABA receptor-gated channels (data not shown).

(d) ASIC Blockade Protects Acidosis-Induced, Glutamate-IndependentNeuronal Injury

FIGS. 8A-C show exemplary data suggesting that acid incubation mayinduce glutamate receptor-independent neuronal injury protected by ASICblockade. FIGS. 8A and 8B show graphs presenting exemplary data fortime-dependent LDH release induced by 1 hr (FIG. 8A) or 24 hr incubation(FIG. 8B) of cortical neurons in pH 7.4 (solid bars) or 6.0 ECF (openbars). N=20-25 wells, *p<0.05, and **p<0.01, compared to pH 7.4 group atthe same time points (acid-induced neuronal injury with fluoresceindiacetate (FDA) also was analyzed by staining of cell bodies of aliveneurons and propidium iodide (PI) staining of nuclei of dead neurons).FIG. 8C shows a graph illustrating inhibition of acid-induced LDHrelease by 100 μM amiloride or 100 ng/mL PcTX venom (n=20-27, *p<0.05,and **p<0.01). MK801, CNQX, and nimodipine were present in ECF for allexperiments (FIGS. 8A-C).

Acid-induced injury was studied on neurons grown on 24-well platesincubated in either pH 7.4 or 6.0 ECF containing MK801, CNQX, andnimodipine; see FIGS. 8A-C. Cell injury was assayed by the measurementof lactate dehydrogenase (LDH) release (Koh and Choi, J. Neurosci.,1987, 20:83-90) at various time points (FIGS. 8A and 8B) and byfluorescent staining of alive/dead cells. Compared to neurons treated atpH 7.4, 1 hr acid incubation (pH 6.0) induced a time-dependent increasein LDH release (FIG. 8A). After 24 hr, 45.7%±5.4% of maximal LDH releasewas induced (n=25 wells). Continuous treatment at pH 6.0 induced greatercell injury (FIG. 8B, n=20). Consistent with the LDH assay, alive/deadstaining with fluorescein diacetate and propidium iodide showed similarincreases in cell death by 1 hr acid treatment (data not shown). Onehour incubation with pH 6.5 ECF also induced significant but less LDHrelease than with pH 6.0 ECF (n=8 wells, data not shown).

The effect of amiloride and PcTX venom on acid-induced LDH release weretested to determine whether activation of ASICs is involved inacid-induced glutamate receptor-independent neuronal injury. Addition ofeither 100 μM amiloride or 100 ng/mL PcTX venom 10 min before and duringthe 1 hr acid incubation significantly reduced LDH release (FIG. 8C). At24 hr, LDH release was decreased from 45.3%±3.8% to 31.1%±2.5% byamiloride and to 27.9%±2.6% by PcTX venom (n=20-27, p<0.01). Addition ofamiloride or PcTX venom in pH 7.4 ECF for 1 hr did not affect baselineLDH release, although prolonged incubation (e.g., 5 hr) with amiloridealone increased LDH release (n=8, data not shown).

(e) Activation of Homomeric ASIC1a is Responsible for Acidosis-InducedInjury

FIGS. 9A-D are a series of graphs presenting exemplary data indicatingthat ASIC1a may be involved in acid-induced injury in vitro. FIG. 9Ashows exemplary data illustrating inhibition of acid-induced LDH releaseby reducing [Ca²⁺]_(e) (n=11-12, **p<0.01 compared with pH 6.0, 1.3Ca²⁺). FIG. 9B shows exemplary data illustrating acid incubation inducedincrease of LDH release in ASIC1a-transfected but not nontransfectedCOS-7 cells (n=8-20). Amiloride (100 μM) inhibited acid-induced LDHrelease in ASIC1a-transfected cells. *p<0.05 for 7.4 versus 6.0 and 6.0versus 6.0+amiloride. FIG. 9C shows exemplary data illustrating a lackof acid-induced injury and protection by amiloride and PcTX venom inASIC1^(−/−) neurons (n=8 in each group, p>0.05). FIG. 9D shows exemplarydata illustrating acid-induced increase of LDH release in culturedcortical neurons under OGD (n=5). LDH release induced by combined 1 hrOGD/acidosis was not inhibited by trolox and L-NAME (n=8-11). OGD didnot potentiate acid-induced LDH release in ASIC1^(−/−) neurons. **p<0.01for pH 7.4 versus pH 6.0 and *p<0.05 for pH 6.0 versus 6.0+PcTX venom.MK801, CNQX, and nimodipine were present in ECF for all experiments(FIGS. 9A-D).

Neurons were treated with pH 6.0 ECF in the presence of normal orreduced [Cal²⁺]_(e) to determine whether Ca²⁺ entry plays a role inacid-induced injury (see FIGS. 9A-D). Reducing Ca²⁺ from 1.3 to 0.2 mMinhibited acid-induced LDH release (from 40.0%±4.1% to 21.9%±2.5%), asdid ASIC1a blockade with PcTX venom (n=11-12, p<0.01; FIG. 9A).Ca²⁺-free solution was not tested, as a complete removal of [Cal²⁺]_(e)may activate large inward currents through a Ca²⁺-sensing cationchannel, which may otherwise complicate data interpretation. Inhibitionof acid injury by both amiloride and PcTX, nonspecific and specificASIC1a blockers, and by reducing [Ca²⁺]_(e) suggests that activation ofCa²⁺-permeable ASIC1a may be involved in acid-induced neuronal injury.

Acid injury of nontransfected and ASIC1a transfected COS-7 cells wasstudied to provide additional evidence that activation of ASIC1a isinvolved in acid injury. COS-7 is a cell line commonly used forexpression of ASICs due to its lack of endogenous channels. Followingconfluence (36-48 hr after plating), cells were treated with either pH7.4 or 6.0 ECF for 1 hr. LDH release was measured 24 hr after acidincubation. Treatment of nontransfected COS-7 cells with pH 6.0 ECF didnot induce increased LDH release when compared with pH 7.4-treated cells(10.3%±0.8% for pH 7.4, and 9.4%±0.7% for pH 6.0, N=19 and 20 wells;p>0.05, FIG. 9B). However, in COS-7 cells stably transfected withASIC1a, 1 hr incubation at pH 6.0 significantly increased LDH releasefrom 15.5%±2.4% to 24.0%±2.9% (n=8 wells, p<0.05). Addition of amiloride(100 μM) inhibited acid-induced LDH release in these cells (FIG. 9B).

Acid injury of CHO cells transiently transfected with cDNAs encoding GFPalone or GFP plus ASIC1a was also studied. After the transfection (24-36hr), cells were incubated with acidic solution (pH 6.0) for 1 hr, andcell injury was assayed 24 hr following the acid incubation. One houracid incubation largely reduced surviving GFP-positive cells inGFP/ASIC1a group but not in the group transfected with GFP alone (datanot shown).

Cell toxicity experiments on cortical neurons cultured from ASIC^(+/+)and ASIC1^(−/−) mice were performed to further demonstrate aninvolvement of ASIC1a in acidosis-induced neuronal injury. Again, 1 hracid incubation of ASIC^(+/+) neurons at 6.0 induced substantial LDHrelease that was reduced by amiloride and PcTX venom (n=8-12). One houracid treatment of ASIC1^(−/−) neurons, however, did not inducesignificant increase in LDH release at 24 hr (13.8%±0.9% for pH 7.4 and14.2%±1.3% for pH 6.0, N=8, p>0.05), indicating resistance of theseneurons to acid injury (FIG. 9C). In addition, knockout of the ASIC1gene also eliminated the effect of amiloride and PcTX venom onacid-induced LDH release (FIG. 9C, n=8 each), further suggesting thatthe inhibition of acid-induced injury of cortical neurons by amilorideand PcTX venom (FIG. 8C) was due to blockade of ASIC1 subunits. Incontrast to acid incubation, 1 hr treatment of ASIC1^(−/−) neurons with1 mM NMDA+10 μM glycine (in Mg²⁺-free [pH 7.4] ECF) induced 84.8%±1.4%of maximal LDH release at 24 hr (n=4, FIG. 9C), indicating normalresponse to other cell injury processes.

(f) Modeled Ischemia Enhances Acidosis-Induced Glutamate-IndependentNeuronal Injury via ASICs

As the magnitude of ASIC currents may be potentiated by cellular andneurochemical components of brain ischemia-cell swelling, arachidonicacid, and lactate and, more importantly, the desensitization of ASICcurrents may be reduced dramatically by modeled ischemia (see FIGS. 5Aand 5C), activation of ASICs in ischemic conditions is expected toproduce greater neuronal injury. To test this hypothesis, neurons weresubjected to 1 hr acid treatment under oxygen and glucose deprivation(OGD). MK801, CNQX, and nimodipine were added to all solutions toinhibit voltage-gated Ca²⁺ channels and glutamate receptor-mediated cellinjury associated with OGD. One hour incubation with pH 7.4 ECF underOGD conditions induced only 27.1%±3.5% of maximal LDH release at 24 hr(n=5, FIG. 9D). This finding is in agreement with a previous report that1 hr OGD does not induce substantial cell injury with the blockade ofglutamate receptors and voltage-gated Ca²⁺ channels (Aarts et al.,2003). However, 1 hr OGD, combined with acidosis (pH 6.0), induced73.9%±4.3% of maximal LDH release (n=5, FIG. 9D, p<0.01), significantlylarger than acid-induced LDH release in the absence of OGD (see FIG. 8A,p<0.05). Addition of the ASIC1a blocker PcTX venom (100 ng/mL)significantly reduced acid/OGD-induced LDH release to 44.3%±5.3% (n=5,p<0.05, FIG. 9D).

The same experiment was performed with cultured neurons from theASIC1^(−/−) mice. Unlike in ASICI containing neurons, however, 1 hrtreatment with combined OGD and acid only slightly increased LDH releasein ASIC1^(−/−) neurons (from 26.1%±2.7% to 30.4%±3.5%, N=10-12, FIG.9D). This finding suggests that potentiation of acid-induced injury byOGD may be due largely to OGD potentiation of ASIC1-mediated toxicity.

It has been demonstrated that activation of a Ca²⁺-permeablenonselective cation conductance activated by reactive oxygen/nitrogenspecies resulting in glutamate receptor-independent neuronal injury(Aarts et al, Cell, 2003, 115:863-877) . The prolonged OGD-induced cellinjury may be reduced dramatically by agents either scavenging freeradicals directly (e.g., trolox) or reducing the production of freeradicals (e.g., L-NAME). To determine whether combined short durationOGD and acidosis induced neuronal injury may involve a similarmechanism, the effect of trolox and L-NAME on OGD/acid-induced LDHrelease was tested. As shown in FIG. 9D, neither trolox (500 μM) norL-NAME (300 μM) had significant effect on combined 1 hrOGD/acidosis-induced neuronal injury (n=8-11). Additional experimentsalso demonstrated that the ASIC blockers amiloride and PcTX venom had noeffect on the conductance of TRPM7 channels (Aarts et al. supra).Together, these findings strongly suggest that activation of ASICs butnot TRPM7 channels may be largely responsible for combined 1 hrOGD/acidosis-induced neuronal injury in our studies.

(g) Activation of ASIC1a in Ischemic Brain Injury In Vivo

FIGS. 10A-D show data illustrating neuroprotection by ASICI blockade andASICI gene knockout in brain ischemia in vivo. FIG. 10A shows a graph ofexemplary data obtained from TTC-stained brain sections illustrating thestained volume (“infarct volume”) in brains from aCSF (n=7), amiloride(n=11), or PcTX venom (n=5) injected rats. *p<0.05 and **p<0.01 comparedwith aCSF injected group. FIG. 10B shows a graph of exemplary dataillustrating reduction in infarct volume in brains from ASIC1^(−/−) mice(n=6 for each group). *p<0.05 and **p<0.01 compared with +/+ group. FIG.10C shows a graph of exemplary data illustrating reduction in infarctvolume in brains from mice i.p. injected with 10 mg/kg memantine (Mem)or i.p. injection of memantine accompanied by i.c.v. injection of PcTXvenom (500 ng/mL). **p<0.01 compared with aCSF injection and betweenmemantine and memantine plus PcTX venom (n=5 in each group). FIG. 10Dshows a graph of exemplary data illustrating reduction in infarct volumein brains from either ASIC1^(+/+) (wt) or ASIC1^(−/−) mice i.p. injectedwith memantine (n=5 in each group). *p<0.05, and **p<0.01.

The protective effect of amiloride and PcTX venom in a rat model oftransient focal ischemia (Longa et al., Stroke, 1989, 20:84-91) wastested to determine whether activation of ASIC1a is involved in ischemicbrain injury in vivo. Ischemia (100 min) was induced by transient middlecerebral artery occlusion (MCAO). A total of 6 μl artificial CSF (aCSF)alone, aCSF-containing amiloride (1 mM), or PcTX venom (500 ng/mL) wasinjected intracerebroventricularly 30 min before and after the ischemia.The volume for cerebral ventricular and spinal cord fluid for 4-week-oldrats is estimated to be ˜60 μl. Assuming that the infused amiloride andPcTX were uniformly distributed in the CSF, a concentration of ˜100 μMfor amiloride and ˜50 ng/mL for PcTX were expected, which is aconcentration found effective in cell culture experiments. Infarctvolume was determined by TTC staining (Bederson et al., Stroke, 1986,17:1304-1308) at 24 hr following ischemia. Ischemia (100 min) producedan infarct volume of 329.5±25.6 mm³ in aCSF-injected rats (n=7) but only229.7±41.1 mm³ in amiloride-injected (n=11, p<0.05) and 130.4±55.0 mm³(˜60% reduction) in PcTX venom-injected rats (n=5, p<0.01) (FIG. 10A).

ASIC1^(−/−) mice were used to further demonstrate the involvement ofASIC1a in ischemic brain injury in vivo. Male ASIC1^(+/+), ASIC1^(+/−),and ASIC1^(−/−) mice (˜25 g, with congenic C57B16 background) weresubjected to 60 min MCAO as previously described (Stenzel-Poore et al.,Lancet, 2003, 362:1028-1037). Consistent with the protection bypharmacological blockade of ASIC1a (above), −/− mice displayedsignificantly smaller (˜61% reduction) infarct volumes (32.9±4.7 mm³,N=6) as compared to +/+ mice (84.6±10.6 mm³, N=6, p<0.01). +/− mice alsoshowed reduced infarct volume (56.9.+−.6.7 mm³, N=6, p<0.05) (FIG. 10B).

To determine whether blockade of ASIC1a channels or knockout of theASIC1 gene would provide additional protection in vivo in the setting ofglutamate receptor blockade, memantine (10 mg/kg) was injectedintraperitoneally (i.p.) into C57B16 mice immediately following 60 minMCAO and accompanied by intracerebroventricular injection (i. c. v.) ofa total volume of 0.4 μl aCSF alone or aCSF containing PcTX venom (500ng/mL) 15 min before and following ischemia. In control mice with i.p.injection of saline and i. c. v. injection of aCSF, 60 min MCAO inducedan infarct volume of 123.6±5.3 mm³ (n=5, FIG. 10C). In mice withintraperitoneal injection of memantine and intracerebroventricularinjection of aCSF, the same duration of ischemia induced an infarctvolume of 73.8.+-.6.9 mm³ (n=5, p<0.01). However, in mice injected withmemantine and PcTX venom, an infarct volume of only 47.0±1.1 mm³ wasinduced (n=5, p<0.01 compared with both control and memantine groups,FIG. 10C). These data suggest that blockade of homomeric ASIC1a mayprovide additional protection in in vivo ischemia in the setting of NMDAreceptor blockade. Additional protection was also observed inASIC1^(−/−) mice treated with pharmacologic NMDA blockade (FIG. 10D). InASIC^(+/+) mice i.p. injected with saline or 10 mg/kg memantine, 60 minMCAO induced an infarct volume of 101.4±9.4 mm³ or 61.6±12.7 mm³,respectively (n=5 in each group, FIG. 10D). However, in ASIC1^(−/−) miceinjected with memantine, the same ischemia duration induced an infarctvolume of 27.7±1.6 mm³ (n=5), significantly smaller than the infarctvolume in ASIC1^(+/+) mice injected with memantine (p<0.05).

Taken together, these data demonstrate that activation of Ca²⁺-permeableASIC1a is a novel, glutamate-independent biological mechanism underlyingischemic brain injury. .

Example 2: Time Window of PcTX Neuroprotection

This example describes exemplary experiments that measure theneuroprotective effect of PcTX venom at different times after onset ofstroke in rodents; see FIG. 11. Briefly, brain ischemia (stroke) wasinduced in rodents by mid-cerebral artery occlusion (MCAO). At theindicated times after induction, artificial cerebrospinal fluid (aCSF),PcTX venom (0.5 μL, 500 ng/mL total protein), or inactivated (boiled)venom was infused into the lateral ventricles of each rodent. As shownin FIG. 11, administration of PcTX venom provided a 60% reduction instroke volume both at one hour and at three hours after stroke onset.Furthermore, substantial stroke volume reduction still may be maintainedif treatment is withheld for five hours after the onset of the MCAO.Accordingly, neuroprotection due to ASIC inhibition may have an extendedtherapeutic time window after stroke onset, allowing stroke subjects tobenefit from treatment performed hours after the stroke began. Thiseffect of ASIC blockade on stroke neuroprotection is far more robustthan that of calcium channel blockade of the NMDA receptor (a majortarget for experimental stroke therapeutics) using a glutamateantagonist. No glutamate antagonist, thus far, has such a favorableprofile as shown here for ASIC1a-selective inhibition.

Example 3: Exemplary Cystine Knot Peptides

This example describes exemplary cystine knot peptides, includingfull-length PcTx1 and deletion derivatives of PcTx, which may bescreened in cultured cells, tested in ischemic animals (e.g., rodentssuch as mice or rats), and/or administered to ischemic human subjects.

FIG. 12 shows the primary amino acid sequence (SEQ ID NO:1), inone-letter code, of an exemplary cystine knot peptide, PcTx1, indicatedat 50, with various exemplary peptide features shown relative to aminoacid positions 1-40. Peptide 50 may include six cysteine residues thatform cystine bonds 52, 54, 56 to create a cystine knot motif 58. Thepeptide also may include one or more beta sheet regions 60 and apositively charged region 62. An N-terminal region 64 and a C-terminalregion 66 may flank the cystine knot motif

FIG. 13 shows a comparison of the PcTx1 peptide 50 of FIG. 12 alignedwith various exemplary deletion derivatives of the peptide. Thesederivatives may include an N-terminal deletion 70 (SEQ ID NO:2), apartial C-terminal deletion 72 (SEQ ID NO:3), a full C-terminal deletion74 (SEQ ID NO:4), and an N/C terminal deletion 76 (SEQ ID NO:5). Otherderivatives of PcTx1 may include any deletion, insertion, orsubstitution of one or more amino acids, for example, while maintainingsequence similarity or identity of at least about 25% or about 50% withthe original PcTx1 sequence.

Each PcTx1 derivative may be tested for its ability to inhibit ASICproteins selectively and/or for an effect, if any, on ischemia. Anysuitable test system(s) may be used to perform this testing includingany of the cell-based assay systems and/or animal model systemsdescribed elsewhere in the present teachings. The PcTx1 derivative alsoor alternatively may be tested in ischemic human subjects.

Example 4: Selectivity of PcTX Venom for ASIC1a

This example describes experiments that measure the selectivity of PcTXvenom (and thus PcTx1 toxin) for ASIC1a alone, relative to other ASICproteins or combinations of ASIC proteins expressed in cultured cells.COS-7 cells expressing the indicated ASIC proteins were treated withPcTX venom (25 ng/mL on ASIC1a expressing cells and 500 ng/mL on ASIC2a,ASIC3 or ASIC1a+2a expressing cells). Channel currents were measured atthe pH of half maximal channel activation (pH 0.5). As shown in FIG. 14,PcTX venom largely blocked the currents mediated by ASIC1a homomericchannels at a protein concentration of 25 ng/mL, with no effect on thecurrents mediated by homomeric ASIC2a, ASIC3, or heteromericASIC1a/ASIC2a at 500 ng/mL (n=3-6). At 500 ng/mL, PcTX venom also didnot affect the currents mediated by other ligand-gated channels (e.g.NMDA and GABA receptor-gated channels) and voltage-gated channels (e.g.Na+, Ca2+, and K+ channels) (n=4-5). These experiments indicate thatPcTX venom and thus PcTx1 peptide is a specific blocker for homomericASIC1a. Using this cell-based assay system, the potency and selectivityof ASIC inhibition may be measured for various synthetic peptides orother candidate inhibitors (e.g., see Example 3).

Example 5: Nasal Administration of PcTX Venom is Neuroprotective

This example describes exemplary data indicating the efficacy of nasallyadministered PcTX venom for reducing ischemia-induced injury in ananimal model system of stroke. Cerebral ischemia was induced in malemice by mid-cerebral artery occlusion. One hour after occlusion wasinitiated animals were treated as controls or were treated with PcTXvenom (50 μL of 5 ng/mL (total protein) PcTx venom introducedintranasally). As shown in FIG. 15, nasal administration of PcTX venomresulted in a 55% reduction in ischemia-induced injury (ischemicdamage), as defined by infarct volume, relative to control treatment.Nasal administration may be via a spray that is deposited substantiallyin the nasal passages rather than inhaled into the lungs and/or may bevia an aerosol that is at least partially inhaled into the lungs. Insome examples, nasal administration may have a number of advantages overother routes of administration, such as more efficient delivery to thebrain and/or adaptability for self-administration by an ischemicsubject.

Example 6: Inhibition of ASIC1a Channel By Amiloride And AmilorideAnalogs

As shown in FIGS. 16A-C, amiloride and amiloride analogs benzamil,phenamil and EIPA block ASIC1a current in a dose-dependent manner.Similarly, amiloride and amiloride analogs benzamil and EIPA blockASIC2a current in a dose-dependent manner (FIGS. 17A-C). Table 1summarizes inhibition of the ASIC1a channel by amiloride and amilorideanalogs. Amiloride was an effective blocker of this channel with an IC50of 7.7 μM.

TABLE 1 Inhibition of the ASIC1a channel by amiloride and amilorideanalogs. Knockout 1a 39.1 ± 3.8 (n = 4) Knockout 2a 5.14 ± 0.79 (n = 5)Neuron (−60 mV) 43.3 ± 1.43 (n = 6) EIPA Neuron (−20 mV) 32.2 ± 6.3 (n =3) CHO 1a 111 ± 30 (n = 5) CHO 2a 31 (n = 1) Knockout 1a 35.9 ± 2.1 (n =5) Knockout 2a 20.1 ± 2.2 (n = 2) Neuron (−60 mV) 82.9 ± 5.2 (n = 8)Bepridil Neuron (−60 mV) 100 ± 11 (n = 10) KB-R7943 Neuron (−60 mV) 24.3± 17.2 (n = 2) 5-(N-Methyl-N-isobutyl)- amiloride Neuron (−60 mV) 15.0 ±11.7 (n = 3) 5-(N,N- hexamethylene)amiloride Neuron (−60 mV) 14.8 ± 7.1(n = 2) 5-(N,N-Dimenthyl)amiloride hydrochloride

Example 7: Reduction of Infarct Volume in Mice byIntracerebroventricular Injection of Amiloride And Amiloride Analogs

Mice were subjected to 60 minutes of middle cerebral artery occlusion(MCAO) as described above. Amiloride or an amiloride analog benzamil,bepridel, EIPA or KB-R7943 was administered by intracerebroventricularinjection one hour after MCAO. The animals were evaluated one day afterischemia induction. As shown in FIG. 18, intracerebroventricularinjection of amiloride or an amiloride analog benzamil, bepridel, EIPAor KB-R7943 effectively reduce infarct volume.

Example 8: Reduction of Infarct Volume in Mice by Intravenous Injectionof Amiloride

Mice were subjected to 60 minutes of middle cerebral artery occlusion(MCAO) as described above. Amiloride was administered by intravenousinjection 1, 3 or 5 hours after MCAO. The animals were evaluated one dayafter ischemia induction. As shown in FIG. 19, intravenous injection ofamiloride effectively reduce infarct volume. The effective CNSpenetration of amiloride may be explained by the fact thatblood-brain-barrier is compromised following brain ischemia/reperfusion.FIG. 20 shows that intravenous injection of amiloride has a prolongedtherapeutic window of 5 h.

Example 9: Structure Activity Relationships for Hydrophobic AmilorideAnalogs on Various Channels

As shown in Table 1, substituting the C-5 amino group in amiloride withalkyl groups led to a decrease in potency at the ASIC1a channel . Thesame substitution increases potency to the ASIC3 channel (Kuduk et al.,Bioorg. Med. Chem. Lett., 2009, 19:2514-2518). The reverse result wasobtained when substituting hydrophobic groups onto the guanidino part ofthe structure. Indeed, the benzyl substituted guanidino analog,benzamil, was the most potent ASIC1a blocking compound tested (IC50=4.9μM). Taken together, these results showed that amiloride is an effectiveblocker of ASUC1a with an IC₅₀ of 7.7 μM. They also provide structureactivity relationships (see FIG. 21) for designing amiloride analogsthat would inhibit the ASIC1a channel. Accordingly, in some embodiments,amiloride analogs are generated by introducing changes in the guanidineportion of the amiloride structure. Since amiloride is only a very weakinhibitor of the Na⁺/Ca²⁺ ion exchanger (IC₅₀=1.1 mM). The amilorideanalogs are likely be a very weak inhibitor of the Na⁺/Ca²⁺ ionexchanger as well. In some embodiments, the amiloride analogs aredesigned to have increased selectivity for ASIC1a over the ASIC3channel. In other embodiments, a ring structure, such as a cyclicguanidine group, is introduced into the amiloride structure to increaseinhibitory potency of ASIC1a currents. It is also possible that one ormore of the N-H groups of amiloride will form H-bonds either internallywith the 3-amino group or with the ion channel.

The in vivo results in mice showed that efficacy can be achieved with aplasma concentration of 32.5 μM (iv dose of 50 μl×1 mM) and a totalbrain concentration of 12.5 μM (icy dose of 1 μl×500 μM). It is thusestimated that only a 10-fold increase in potency is necessary toachieve efficacious concentrations that are suitable for an acutetherapeutic for stroke in humans. Therefore, novel analogs are screenedfor an increased ASIC1a IC50 potency from the 4 to 8 μM for amilorideand benzamil to <1 μM.

In some embodiments, the amiloride analogs comprise methylated analogsof benzamil (formula 1-5 of FIG. 21) and amidino analog of benzamil(formula 6 of FIG. 21). In other embodiments, the amiloride analogscontain a ring formed on the guanidine group. In other embodiments, theamiloride analogs contain an acylguanidino group for increasedinhibitory potency of ASIC1a currents.

Amiloride is soluble in water at 1 mM and is effective in treatingischemia in a mouse model at a dose of 50 ul per injection. Theequivalent dose on a mg/kg basis in a 65 kg human would be close to 40mg and require an injection volume of over 160 ml. Similarly benzamilhas a reported solubility in 0.9% saline of 0.4 mg/ml (1.7 mM), whichpermits administration of only 5 mg benzamil dihydrochloride in a 10 mlinjection. Accordingly, amiloride analogs with higher water solubilityare desired. In some embodiments, the amiloride analogs contain a watersolubilizing group, such as an N,N-dimethyl amino group or a sugar, atthe guanidino group to improve water solubility. In some embodiments,the amiloride analogs have a water solubility of 5 mM, 10 mM, 20 mM, 30mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or higher. In otherembodiments, the amiloride analogs have a solubility that allows for a10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or500 mg dose to be administered intravenously to a human in a single 10ml injection. In yet other embodiments, the amiloride analogs have asolubility that allows for a 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200mg, 250 mg, 300 mg, 400 mg, or 500 mg dose to be administeredintracerebroventicularly to a human in a single 2 ml injection.

The disclosure set forth above may encompass one or more distinctinventions, with independent utility. Each of these inventions has beendisclosed in its preferred form(s). These preferred forms, including thespecific embodiments thereof as disclosed and illustrated herein, arenot intended to be considered in a limiting sense, because numerousvariations are possible. The subject matter of the inventions includesall novel and nonobvious combinations and subcombinations of the variouselements, features, functions, and/or properties disclosed herein. Thefollowing claims particularly point out certain combinations andsubcombinations regarded as novel and nonobvious. Inventions embodied inother combinations and subcombinations of features, functions, elements,and/or properties may be claimed in applications claiming priority fromthis or a related application. Such claims, whether directed to adifferent invention or to the same invention, and whether broader,narrower, equal, or different in scope to the original claims, also areregarded as included within the subject matter of the inventions of thepresent disclosure.

1. A method for reducing nerve injury in an adult human subject,comprising: administering to said subject a therapeutically effectiveamount of a pharmaceutical composition comprising amiloride, anamiloride analog, or a pharmaceutically acceptable salt thereof, whereinthe pharmaceutical composition is administered intravenously and whereinthe nerve injury is acidosis-induced nerve injury.
 2. The method ofclaim 1, wherein said active ingredient comprises amiloride or apharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein said active ingredient comprises an amiloride analog or apharmaceutically acceptable salt thereof.
 4. The method of claim 3,wherein said amiloride analog is selected from the group consisting ofbenzamil, phemnil, 5-(N-ethyl-N-isobutyl)-amiloride (EIPA), bepridil,KB-R7943, 5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimenthyl) amiloride hydrochloride.
 5. The methodof claim 4, wherein said amiloride analog is benzamil.
 6. The method ofclaim 3, wherein said amiloride analog is a methylated analog of benzami7. The method of claim 3, wherein said amiloride analog comprises a ringformed on a guanidine group.
 8. The method of claim 3, wherein saidamiloride analog comprises an acylguanidino group.
 9. The method ofclaim 3, wherein said amiloride analog comprises a water solubilizinggroup formed on a guanidine group, wherein the water solubilizing groupis a N,N-dimethyl amino group or a sugar group. 10-12. (canceled) 13.The method of claim 2, wherein said amiloride, amiloride analog or apharmaceutically acceptable salt thereof is given in a dose range of 0.1mg-10 mg/kg body weight. 14-16. (canceled)
 17. The method of claim 1,wherein said nerve injury is a nervous system injury.
 18. The method ofclaim 1, wherein said nerve injury is brain injury. 19-42. (canceled)